The Role of the Plant-Specific ALTERED XYLOGLUCAN9 Protein in Arabidopsis Cell Wall Polysaccharide O-Acetylation

A mutation in the ALTERED XYLOGLUCAN9 (AXY9) gene was found to be causative for the decreased xyloglucan acetylation phenotype of the axy9.1 mutant, which was identified in a forward genetic screen for Arabidopsis (Arabidopsis thaliana) mutants. The axy9.1 mutant also exhibits decreased O-acetylation of xylan, implying that the AXY9 protein has a broad role in polysaccharide acetylation. An axy9 insertional mutant exhibits severe growth defects and collapsed xylem, demonstrating the importance of wall polysaccharide O-acetylation for normal plant growth and development. Localization and topological experiments indicate that the active site of the AXY9 protein resides within the Golgi lumen. The AXY9 protein appears to be a component of the plant cell wall polysaccharide acetylation pathway, which also includes the REDUCED WALL ACETYLATION and TRICHOME BIREFRINGENCE-LIKE proteins. The AXY9 protein is distinct from the TRICHOME BIREFRINGENCE-LIKE proteins, reported to be polysaccharide acetyltransferases, but does share homology with them and other acetyltransferases, suggesting that the AXY9 protein may act to produce an acetylated intermediate that is part of the O-acetylation pathway.The plant cell wall is a complex composite of polysaccharides, glycoproteins, and polyphenols, with the fine structure and quantity of each varying by species, tissue, and developmental time point (Knox, 2008; Burton et al., 2010). Cellulose, hemicelluloses, and pectic polysaccharides are the three major classes of polysaccharides observed in the wall. Current models of the wall have cellulose microfibrils as the major structural component, with hemicelluloses binding to the microfibrils and pectins as an amorphous matrix in which the cellulose/hemicellulose network is embedded (Pauly et al., 1999a; Somerville et al., 2004; Cosgrove, 2005). Unlike the linear β-1,4-glucan chains making up cellulose microfibrils, hemicelluloses and pectins consist of a diverse set of glycosyl units and linkages as well as other modifications such as methylation and acetylation (Caffall and Mohnen, 2009; Scheller and Ulvskov, 2010; Pauly et al., 2013).The O-acetyl substitutions on hemicelluloses and pectins occur on a variety of specific glycosyl residues. The hemicellulose xyloglucan (XyG) consists of a β-1,4-glucan backbone with a regular pattern of xylosyl branches, with additional galactosyl, fucosyl, arabinosyl, and/or galacturonosyl substitution depending on the tissue and plant species (Obel et al., 2009; Pauly et al., 2013; Schultink et al., 2014). XyG O-acetylation has been reported on the β-1,4-glucan backbone (Sims et al., 1996; York et al., 1996) as well as on specific galactosyl or arabinosyl side chains (Kiefer et al., 1989; Vierhuis et al., 2001). The hemicellulose xylan is heavily acetylated at positions O2 and O3 of the backbone β-1,4-xylosyl residues, with the degree of acetylation (O-acetyl groups per backbone of xylosyl residue) ranging from approximately 0.4 to 0.6 depending on the species (Teleman et al., 2002; Evtuguin et al., 2003; Prozil et al., 2012; Chong et al., 2014; Lee et al., 2014). The glycosyl substituents of xylan, including glucuronosyl, arabinosyl, and xylosyl groups, have not been reported to be acetylated. The backbone β-1,4-mannosyl residues of the hemicellulosic polysaccharide mannan also can be acetylated (Manna and McAnalley, 1993). The predominant location of O-acetyl groups in pectin has been reported to be on galacturonic acid residues at positions O2 and O3 (Ralet et al., 2005). O-Acetylation of pectin also has been observed on rhamnosyl (Sengkhamparn et al., 2009), fucosyl, and aceric acid residues (Glushka et al., 2003).The functional significance and biosynthetic pathway of wall polysaccharide O-acetylation are not fully understood. O-Acetylation has been shown to influence the solubility, gelation, and enzymatic accessibility of polysaccharides in vitro (Biely et al., 1986; Huang et al., 2002). These properties are likely to be important for appropriate function in planta. Recently identified Arabidopsis (Arabidopsis thaliana) mutants with polysaccharide O-acetylation deficiencies (reduced wall acetylation [rwa] and trichome birefringence-like [tbl]; Gille and Pauly, 2012) have allowed for testing of the in vivo role of this substituent. The ALTERED XYLOGLUCAN4 (AXY4 [TBL27]) gene from the TBL family was identified in a forward genetic screen of Arabidopsis and is believed to code for a XyG acetyltransferase (Gille et al., 2011). The growth morphology of this mutant, which lacks XyG O-acetylation in leaves, etiolated seedlings, and roots, was not affected under laboratory growth conditions. Arabidopsis mutants deficient for a putative xylan acetyltransferase (TBL29/ESKIMO1 [ESK1]) were reported to have reduced growth and irregular xylem and to be freezing tolerant (Xin et al., 2007; Xiong et al., 2013; Yuan et al., 2013). Arabidopsis mutants deficient for other TBL genes have been reported to exhibit phenotypes such as aberrant trichomes (Bischoff et al., 2010a) and resistance to powdery mildew (Vogel et al., 2004), but polysaccharide acetylation defects have not been demonstrated in these cases. The variation in the morphological phenotypes of different tbl mutants suggests that the function of polysaccharide acetylation is specific to the particular polysaccharide and tissue.While the TBL gene products seem to affect single wall polysaccharides, Arabidopsis mutants defective for one or more of the four RWA genes have decreased acetylation of multiple polysaccharides and growth phenotypes ranging from mild to severe (Lee et al., 2011; Manabe et al., 2011, 2013). For this reason, and because the RWA proteins are integral membrane proteins with 10 predicted transmembrane domains, it has been hypothesized that they may act as transporters for an activated form of acetate into the Golgi apparatus (Manabe et al., 2011). It has been demonstrated that acetyl-CoA is involved in the pathway of pectin acetylation (Pauly and Scheller, 2000); however, it is not clear if acetyl-CoA is transported into the Golgi or there is an alternative donor substrate that acts as a carrier.In this study, we report the identification and characterization of AXY9, an additional component of the plant cell wall polysaccharide acetylation pathway.     

Allergic diseases and asthma in the family predict the persistence and onset-age of asthma: a prospective cohort study

BackgroundFamily history of asthma and other allergic diseases have been linked to the risk of childhood asthma previously, but little is known about their effect on the age-of-onset and persistency of asthma until young adulthood.MethodsWe assessed the effect of the family history of asthma and allergic diseases on persistent vs. transient, and early- vs. late-onset persistent asthma in The Espoo Cohort Study 1991–2011, a population-based cohort study of 1623 subjects (follow-up rate 63.2%). The determinants were any family history (any parent or sibling); maternal; paternal; siblings only; parents only; and both siblings and parents. Analyses were conducted separately for asthma and allergic diseases while taking the other disease into account as a confounding factor. The outcomes were persistent, transient, early-onset persistent (<13 years) and late-onset persistent asthma. Adjusted risk ratios (RR) were calculated applying Poisson regression. Q-statistics were used to assess heterogeneity between RRs.ResultsFamily history was associated with the different subtypes but the magnitude of effect varied quantitatively. Any family history of asthma was a stronger determinant of persistent (adjusted RR = 2.82, 95% CI 1.99-4.00) than transient asthma (1.65, 1.03-2.65) (heterogeneity: P = 0.07) and on early-onset than late-onset persistent asthma. Also any family history of allergic diseases was a stronger determinant of persistent and early-onset asthma. The impact of paternal asthma continued to young adulthood (early-onset: 3.33, 1.57-7.06 vs. late-onset 2.04, 0.75-5.52) while the influence of maternal asthma decreased with age (Early-onset 3.94, 2.11-7.36 vs. Late-onset 0.88, 0.28-2.81). Paternal allergic diseases did not follow the pattern of paternal asthma, since they showed no association with late-onset asthma. Also the effect estimates for other subtypes were lower than in other hereditary groups (persistent 1.29, 0.75-2.22 vs. transient 1.20, 0.67-2.15 and early-onset 1.86, 0.95-3.64 vs. late-onset 0.64, 0.22-1.80).ConclusionsFamily history of asthma and allergic diseases are strong determinants of asthma, but the magnitude of effect varies according to the hereditary group so that some subtypes have a stronger hereditary component, and others may be more strongly related to environmental exposures. Our results provide useful information for assessing the prognosis of asthma based on a thorough family history.     

Baseline in vitro efficacy of ACT component drugs on Plasmodium falciparum clinical isolates from Mali

In vitro susceptibility to antimalarial drugs of Malian Plasmodium falciparum isolates collected between 2004 and 2006 was studied. Susceptibility to chloroquine and to three artemisinin-based combination therapy (ACT) component drugs was assessed as a first, to our knowledge, in vitro susceptibility study in Mali. Overall 96 Malian isolates (51 from around Bamako and 45 collected from French travellers returning from Mali) were cultivated in a CO₂ incubator. Fifty percent inhibitory concentrations (IC₅₀s) were measured by either hypoxanthine incorporation or Plasmodium lactate dehydrogenase (pLDH) ELISA. Although the two sets of data were generated with different methods, the global IC₅₀ distributions showed parallel trends. A good concordance of resistance phenotype with pfcrt 76T mutant genotype was found within the sets of clinical isolates tested. We confirm a high prevalence of P. falciparum in vitro resistance to chloroquine in Mali (60–69%). While some isolates showed IC₅₀s close to the cut-off for resistance to monodesethylamodiaquine, no decreased susceptibility to dihydroartemisinin or lumefantrine was detected. This study provides baseline data for P. falciparum in vitro susceptibility to ACT component drugs in Mali.     

Evaluation of the viability and germination of tempisque [<i>Sideroxylon capiri</i> (A.DC.) Pittier Sapotaceae]

The tempisque (Sideroxylon capiri) is a tree native to Mexico used by the rural population for housing construction, poles and hedges, as fuel (wood) and also for fodder and ornamental purposes, among others. It is considered an endangered species. In order to contribute to its preservation and sustainable management, it was considered important to determine the proportion of viable seeds, the loss of viability due to storage period and the germination process by applying pregerminative treatments. We found that freshly collected seeds showed 100% viability, which decreased to 0% after 5 months of storage. According to the cumulative germination significant differences between treatments (p≤0.01) were found. It was observed that seeds can accelerate their time of germination with the previous exposure of 24 h in water at room temperature. The soaking treatment in water for 24 h at room temperature obtained final germination of 55%, while with the control 39% was reached. Soaking in hydrogen peroxide and scarification were the treatments with lower germination percentage (33 and 23%, respectively). To get a higher percentage of germinated seeds in a short time, it is necessary to give a soaking treatment in water for 24 h before sowing.     

Influence of aflatoxin B1 on seed germination, seedling growth, chlorophyll and carotenoid contents of mustard (brassica juncea L. var. pusa bold) seeds

Five different concentrations (100, 250, 500, 1000 and 2000 μg/L of aflatoxin B₁ were found to be inhibitory to seed germination and seedling growth (root and shoot lengths) of mustard seeds (variety Pusa bold). These also lowered the levels of chlorophyll and carotenoids in the emerging leaves during seedling growth. The inhibitory effect was correlated with the concentration of applied toxin.     

Fungi hijack a ubiquitous plant apoplastic endoglucanase to release a ROS scavenging β-glucan decasaccharide to subvert immune responses

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner insoluble cell wall layer and an outer soluble extracellular polysaccharide (EPS) matrix. Here, we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, and hence likely have different biological functions. The barley (Hordeum vulgare) β-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved β-1,3;1,6-glucan decasaccharide (β-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight β-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by β-1,3-endoglucanases due to the presence of three β-1,6-linked glucose branches and can scavenge reactive oxygen species. Exogenous application of β-GD leads to enhanced fungal colonization in barley, confirming its role in the fungal counter-defensive strategy to subvert host immunity. Our data highlight the hitherto undescribed capacity of this often-overlooked EPS matrix from plant-associated fungi to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant–microbe interface.

A β-1,3;1,6-glucan decasaccharide released from the fungal matrix by an apoplastic host hydrolase contributes to plant immune suppression and fungal accommodation.

IN A NUTSHELL Background: Plants secrete various hydrolytic enzymes into the apoplastic space to protect themselves against invading microbes. Some of these enzymes target the fungal cell wall polymer chitin. This enzymatic attack leads to the release of chitin oligomers, which can be perceived by the plant immune system, informing the plant to activate its defense machinery. However, chitin accounts for only a small part of most fungal cell walls. Recent studies have highlighted a largely uncharacterized, β-glucan-rich extracellular polysaccharide matrix (EPS) surrounding the cell wall of various plant-colonizing fungi. Question: This EPS matrix is made of glucose and abundantly produced during colonization. As its secretion into the extracellular environment is costly for the fungus, we explored how this EPS matrix affects plant immunity and fungal colonization. Findings: We demonstrated that EPS matrices from a symbiotic and pathogenic plant-colonizing fungus are distinct from the nonsoluble fungal cell walls with respect to their protein and carbohydrate composition. Enzymatic digests revealed that a secreted plant hydrolase from barley (HvBGLUII) acts on these EPS matrices and releases a highly branched β-glucan decasaccharide (β-GD) fragment. This fragment is not perceived by the plant immune system but instead detoxifies reactive oxygen species produced by the plant host as a defense mechanism and contributes to host colonization. We thus have shown that the outermost fungal EPS layer represents a protective shield against oxidative stress. Next steps: The diversity of linkage types and branching patterns of β-glucans not only accounts for their different biochemical properties, but also makes them important messengers for the plant, potentially encoding specific information on the approaching fungal invader. Future studies should aim to identify other plant hydrolases and the elusive glucan receptors, to disentangle the contribution of β-glucans to the communication between plant hosts and fungi.     

Characterization and localization of myosin in the brush border of intestinal epithelial cells

The brush border of intestinal epithelial cells consists of a tightly packed array of microvilli, each of which contains a core of actin filaments. It has been postulated that microvillar movements are mediated by myosin interactions in the terminal web with the basal ends of these actin cores (Mooseker, M.S. 1976. J. Cell. Biol. 71:417-433). We report here that two predictions of this model are correct: (a) The brush border contains myosin, and (b) myosin is located in the terminal web. Myosin is isolated in 70 percent purity by solubilization of Triton-treated brush borders in 0.6 M KI, and separation of the components by gel filtration. Most of the remaining contaminants can be removed by precipitation of the myosin at low ionic strength. This yield is approximately 1 mg of myosin/30 mg of solubilized brush border protein. The molecule consists of three subunits with molecular weights of 200,000, 19,000, and 17,000 daltons in a 1:1:1 M ratio. At low ionic strength, the myosin forms small, bipolar filaments with dimensions of 300 X 11nm, that are similar to filaments seen previously in the terminal web of isolated brush borders. Like that of other vertebrate, nonmuscle myosins, the ATPase activity of isolated brush border myosin in 0.6 M KCI is highest with EDTA (1 μmol P(i)/mg-min; 37 degrees C), intermediate with Ca++ (0.4 μmol P(i)/mg-min), and low with Mg++ (0.01 μmol P(i)/mg-min). Actin does not stimulate the Mg-ATPase activity of the isolated enzyme. Antibodies against the rod fragment of human platelet myosin cross-react by immunodiffusion with brush border myosin. Staining of isolated mouse or chicken brush borders with rhodamine-antimyosin demonstrates that myosin is localized exclusively in the terminal web.     

Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.